Clear vision requires an optically transparent cornea. Transparency is dependent on corneal hydration, which is controlled by an epithelial cell layer called the corneal endothelium. The endothelium controls hydration by secreting fluid from the corneal stoma to the aqueous humor by ion coupled fluid transport. The membrane and cellular regulatory mechanisms of transport in the endothelium are ot understood. The PI's goal is to develop a model for endothelial fluid transport that includes: the specific membrane transport proteins involved, their polarity of transport, and how transport can be regulated. A long-term goal is to udnerstand how disease, (e.g. fuchs' Endothelial Dystrophy, Diabetes) alters endothelial function. Conceivable, medical therapies can be developed to delay or supplant the need for corneal transplantation in cases of endothelial disease or trauma. Since endothelial transport requires HC03 and Cl, we will examine the mechanisms by which endothelial cells regulate their intracellular pH (pHi), control intracellular (C1) and C1 transport rates, using ion sensitive fluorescent probes for pH, Na+, C1 and Ca2+. Transporter polarity will be determine by: a) physiological measurements on cells grown on permeable filters, b) light and elctron microscope immunocytochemistry and c) western blotting of apical/basolateral membrane fractions. Carbonic anhydrase inhibitors slow endothelial fluid transport. Therefore we will examine the roles of cytoplasmic and membrane bound carbonic anhydrase in HC03 to C02. This will be examined by measuring the effects of HC03 transport and carbonic anhydrase blockers on the unstirred layer pH and fluid transport. Because fluid transport is HC03 dependent, the role of pHi in determining fluid transport will be examined. These studies will provide the basis for future development of medical therapies for restoring the loss of vision that can accompany endothelial disease or trauma.